Interactions of Nonprotic Organic Solvents with [Val5]angiotensin in

B , 2011, 115 (7), pp 1712–1719. DOI: 10.1021/jp1107125. Publication Date (Web): February 2, 2011. Copyright © 2011 American Chemical Society. *E-m...
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Interactions of Nonprotic Organic Solvents with [Val5]angiotensin in Water Robert C. Neuman, Jr. and John T. Gerig* Department of Chemistry and Biochemistry, University of California Santa Barbara, Santa Barbara, California 93106, United States

bS Supporting Information ABSTRACT: Intermolecular solvent-solute nuclear Overhauser effects have been used to explore interactions of the organic component of acetonitrile-water, acetonewater, and dimethyl sulfoxide-water mixtures with the peptide hormone [val5]angiotensin. As reported by the NOEs, many cross relaxation terms for interactions of these organic cosolvents are adequately accounted for using a hard spheres interaction model in which encounters of peptide and cosolvent molecules take place by mutual diffusion. However, there are indications of localized solvent-peptide interactions that are not well described by this model. In dimethyl sulfoxide-water at 0 °C, organic solvent near the C-terminal Phe8 residue and the Val3 residue produce strongly enhanced cross-relaxation terms. NOEs for all peptide N-H protons and the protons of the Tyr4 aromatic ring were significantly more positive than expected in 33% acetone-water (v/v) at 0 °C, while those for most side-chain protons were close to predictions of the hard sphere model. All peptide-organic solvent NOEs in 35% acetonitrile water (v/v) at 0 °C are consistent with the hard spheres interaction model.

’ INTRODUCTION Mixtures of small organic molecules with water are routinely used throughout experimental peptide chemistry. The organic component may simply be needed to increase solubility. Cosolvents such as acetonitrile are used to alter the chromatographic or electrophoretic mobility of peptides.1 Organic cosolvents can alter the conformation, solubility, stability, and propensity for fibril formation of peptides.2-6 Dimethyl sulfoxide (DMSO), for example, is thought to disrupt secondary structure of polypeptides by hydrogen bonding.7 The presence of organic solvents may lead to changes in peptide and protein dynamics.8 In most situations, possible direct interactions of the organic cosolvent with the biological material under study tend to be ignored, although identification of solventbinding sites for small organic molecules can provide useful clues for development of pharmaceuticals.9 In this work, we examine interactions of three commonly used nonprotic organic cosolvents with the peptide [val5]angiotensin II in aqueous solutions. This octapeptide is an analogue of the hormone angiotensin II in which valine at position 5 replaces the usual isoleucine residue. The analogue has been found to have the same biological and immunoreactive properties as endogenous angiotensin II.10 The primary tool has been determination of intermolecular solvent-solute NOEs.11-13 The results provide evidence for selective interactions of the organic cosolvent with parts of the peptide structure. H3 Nþ -Asp1 -Arg2 -Val3 -Tyr4 -Val5 -His6 -Pro7 -Phe8 -COO½val5 angiotensin II

’ EXPERIMENTAL SECTION Materials. [Val5]angiotensin II was obtained in >95% purity

from Sigma-Aldrich or as a greater than 98% purity product from r 2011 American Chemical Society

GenWay (San Diego, CA). Both samples were used as received. Mass spectra were consistent with the expected structure. An impurity with a proton chemical shift of 2.05 ppm, assumed to arise from acetic acid/acetate ion, was observed in the proton NMR spectrum of the Sigma-Aldrich sample. Acetone, acetonitrile, dimethyl sulfoxide, and deuterium oxide (100 atom %) were the highest quality available from Sigma-Aldrich and were used as received. 3-(Trimethylsilyl)propionic acid-d4 sodium salt (TSP) was obtained from Stohler Isotope Chemicals. Distilled, deionized water was used for sample preparation. Sample Preparation. Solvent mixtures were prepared on a v/v basis with the water portion being a mixture of 85% H2O/ 15% D2O. Samples for NMR experiments were 10-15 mM in peptide and contained a trace of TSP to provide a reference signal which was set at 0.0 ppm. The apparent sample pH (3-4) was determined using a Model IQ150 pH meter (IQ Instruments, San Diego, CA) equipped with a 4 mm o.d. stainless steel electrode. The reported pH values are meter readings and were not corrected for the presence of organic solvent or D2O. Samples were degassed by several freeze-thaw cycles before being sealed under vacuo in 5 mm J. Young tubes (Wilmad). NMR Spectroscopy. Carbon-13 decoupled proton spectra were collected at 500 MHz using a Varian INOVA instrument equipped with a Nalorac triple resonance/triple PFG probe. The signal from D2O present in the sample mixture was used as a lock signal. Sample temperatures were determined using a standard sample of methanol (Wilmad) and are believed to have been constant to better than (0.1 °C during the course of an experiment and accurate to better than (0.5 °C. Received: November 9, 2010 Revised: January 7, 2011 Published: February 2, 2011 1712

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The Journal of Physical Chemistry B Pulse sequences for the solvent-peptide NOE experiments involved suppression of both water (OH) and organic solvent (CH3) signals during detection and were based on those published by Dalvit.14 A DPFG-SE sequence15 with 15 Hz bandwidth square pulses was used to achieve selective inversion of the organic solvent resonance at the start of the mixing time rather than the method used by Dalvit.15,16 Selective rf pulses were generated with the Pbox function of the Varian software. Broadband carbon-13 decoupling was present during the application of any selective RF pulse and during fid acquisition. Magic angle gradients were used during the detection phase of all experiments.17 Radiation damping was controlled by imposing a weak field gradient during delays in the pulse sequences. Proton T1 relaxation times were determined by the nonselective inversion-recovery method.18 Self-diffusion coefficients for sample components were determined by bipolar-double-stimulated-echo pulsed field gradient experiments.19 Samples were allowed to equilibrate in the probe at the regulated temperature at least 3 h before diffusion measurements were started. Field gradient pulses were calibrated using the known diffusion coefficient of water in deuterium oxide.20 The reproducibility of the diffusion coefficients suggested that the values given below are reliable to at least (15%. The conclusions reached in this paper are not dependent on high accuracy for the diffusion coefficients and it did not seem profitable to strive for greater accuracy. The initial slopes of NOE vs mixing time curves were determined using the procedures previously described and discussed further in the Supporting Information.18,21-23 Peak intensities were measured for a range of mixing times (50 < tmix < 600 ms) and fit to the empirical function A*tmix þ B*tmix2. The coefficient A was taken as the initial slope of the data. In the absence of any perturbation of the peptide resonances by inversion of the solvent resonance or spin diffusion effects, these initial slopes are equal to the intermolecular cross relaxation term 23 σNOE solvent defined below. Corrections for incomplete inversion of the solvent signal were applied as indicated in the Supporting Information. It was estimated that initial slope parameters have accuracies of (20-50%, depending on the S/N ratio of the signal under consideration. While the DPFG-SE sequence for inversion of the solvent line is highly selective and peptide magnetizations 0.1 ppm away from the solvent resonance appear to be not appreciably influenced by it, solvent-derived Overhauser effects are small and even minor perturbations of peptide starting magnetizations can influence initial slopes of Overhauser effect vs mixing time curves through intramolecular NOEs and spin-lattice relaxation. To examine the influences of solvent inversion on nearby peptide resonances, application of the DPFG-SE sequence was studied with a program based on the Bloch equations.24 Ten or more conformations of the peptide, determined to be consistent with observed intramolecular NOEs as described below, were examined in these studies for each solvent system. Except for the instances noted later, such calculations indicated that initial slopes for the intermolecular NOE experiments would be altered by at most a few percent by any perturbations of peptide magnetizations concomitant with inversion of the solvent magnetization. Data Analysis and Calculations. Molecular modeling and visualizations were done with SYBYL8.0 (Tripos), PYmol (DeLano Scientific LLC), and MOLMOL.25 The program CYANA was used to find conformations consistent with distance

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constraints derived from observed proton-proton NOEs and JCRHNH coupling constants.26,27 Interpretations of solvent NOE data rest on comparing experimental observations of the dependence of an Overhauser effect on mixing time to NOEs predicted by a computational model of the system. The procedures employed were the same as those which have been described elsewhere.21,28 A “full-relaxation matrix” treatment of intramolecular 1H-1H interactions was used.29,30 Values for an effective overall rotational correlation time (τR) and a correlation time for methyl group rotation (τM) for the peptide were needed for this treatment of intramolecular dipolar interactions. The correlation times τR and τM were estimated by comparing peptide proton T1 relaxation behavior calculated using this approach to experimental results. Using a hard sphere interaction model for peptide protonsolvent proton interactions, the intermolecular cross relaxation rate σNOE solvent is given by

3

σ NOE solvent ¼

1 4 2 γ h ½6J2 ð2ωH Þ-J2 ð0Þ 10 H

where J2 is a spectral density function given by Ayant et al.31 It depends on NS, the number of inverted solvent spins per mL and the value of τ = r2/D, where D is sum of the diffusion coefficients for spheres representing the peptide and the solvent spins (D = Dpeptide þ Dsolvent) and r is the distance of their closest approach (r = rpeptide þ rsolvent), with rpeptide and rsolvent being the radii of the representative spheres for a peptide hydrogen and for a solvent molecule, respectively. Because solvent molecules presumably rotate rapidly, all hydrogen atoms of these molecules were assumed to be located at the center of their corresponding representative sphere.32 Radii of spheres representing acetone, acetonitrile, and dimethyl sulfoxide molecules were taken to be 2.56, 2.28, and 2.69 Å, respectively, values estimated using the approach described previously.23 To facilitate comparison of effects between different solvent systems, we define a reduced cross relaxation parameter σR σR ¼

σNOE solvent NS

Although independent of the concentration of the organic solvent, σR depends in a complex way on the diffusion of peptide and solvent species (D) and their interaction distance (r).

’ RESULTS Structural Studies. Proton NMR data for [val5]angiotensin

in various solvent-water mixtures were collected at a sample temperature of 0 °C since previous work had suggested that unanticipated organic cosolvent interactions with the peptide would most likely be observed at temperatures below room temperature.33,34 As was the case in earlier studies, signals were observed for a minor conformation (∼10-20%) of the peptide arising from rotational isomerism at the His6-Pro7 peptide bond. These were ignored in the present work. Proton spectra for the major conformation were assigned using TOCSY, NOESY, and ROESY data. Assigned chemical shifts for all systems are given in the Supporting Information. Vicinal coupling constants (3JNHCRH) observed for the backbone protons of [val5]angiotensin ranged from 7.0 to 10.3 Hz and are given in the Supporting Information. For the peptide dissolved in the three organic solvent-water mixtures used in the 1713

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Table 1. Translational Diffusion Coefficients (106 cm2 s-1) at 0 °C

[val5]angiotensin organic solvent acetate TSP a

33%

35%

36% dimethyl

acetone-water

acetonitrile-water

sulfoxide-water

0.65 2.92

1.01 7.28

0.45 1.60

a

a

2.67

1.95

3.39

1.44

An acetate (impurity) signal not detected in these samples.

Figure 1. Representation of 10 structures of [val5]angiotensin in 33% acetone-water (v/v) consistent with 64 observed intramolecular NOEs. The N-termini are to the left. The mean global rmsd of the backbone atoms, calculated with MOLMOL,25 was 2.4 Å while the rmsd of all heavy atoms was 3.6 Å.

present work, these backbone coupling constants averaged ∼9 Hz, somewhat larger than the average 3JNHCRH ∼ 8 Hz found for the peptide dissolved in several alcohol-water mixtures.21 The observed coupling constants are consistent with the peptide being in largely extended conformations.35 Observed intramolecular proton-proton NOEs and ROEs and the vicinal coupling constants were used in attempts to define the conformation of the peptide in each solvent mixture. The number of NOEs detected depended on the cosolvent; the intramolecular NOEs observed in each system are given in the Supporting Information. The program CYANA was used to develop peptide conformations consistent with the interproton distances indicated by intramolecular 1H-1H Overhauser effects.26,27 The conformational energies of 10 or more of structures so obtained were minimized in the AMBER99 force field.36 The resulting structures obtained for [val5]angiotensin in 33% acetone-water, shown in Figure 1, are typical. Similar results were obtained with the other solvent systems examined and are given in the Supporting Information. As was the case in earlier studies of [val5]angiotensin in alcohol-water systems,21,33,34 the number and distribution of the available distance constraints did not indicate a single dominant structure for the peptide but were found to be consistent with a large number of somewhat folded conformations. Translational Diffusion. Translational diffusion coefficients were measured for the components of samples studied. The results are given in Table 1. Translational diffusion of [val5]angiotensin is sensitive to the hydrodynamic size and shape of the peptide.37-40 The diffusion constant (Dtrans) is typically related to molecular dimensions by means of the Stokes-Einstein equation. This equation is most readily applied to estimating molecular sizes by comparing the diffusion constant of the species of interest to that of a reference material of known dimensions present in the same solution. Thus, evaluating the ratio of diffusion constants provides the hydrodynamic radius of the peptide relative to that of a known reference material: rPeptide DReference trans ¼ Peptide r Reference Dtrans Hydrodynamic radii of [val5]angiotensin were estimated using acetate and TSP as references. The assumed radii of the reference species were 2.2641 and 3.62 Å,42 respectively. The estimated peptide radius was the same within experimental

Figure 2. Intermolecular NOEs on low-field signals of [val5]angiotensin resulting from inversion of the solvent methyl resonance in a mixture of 35% CH3CN-water (v/v) at 0 °C (top). A control spectrum is shown at the bottom. The mixing time for the NOE experiment was 500 ms. NOEs on all upfield signals of the peptide were positive.

uncertainty for all solvent systems, 12 ( 1 Å. This result is close to the average hydrodynamic radius (11 ( 1 Å), estimated in the same way, for the peptide dissolved in a set of alcohol-water solvents.21 The effective size of [val5]angiotensin appears to vary little in going from a protic cosolvent-water to a nonprotic cosolvent-water mixture. Radii of the various peptide conformations found to be consistent with experimental 1H-1H intramolecular NOE data were estimated using the method previously described.23 These calculated radii averaged 7.8 Å, similar to radii calculated for conformations of [val5]angiotensin in several alcohol-water mixtures at 0 °C (7.7 Å).21 35% Acetonitrile at 0 °C. The low-field portion of the proton NMR spectrum of [val5]angiotensin II dissolved in 35% acetonitrile/water v/v is shown in Figure 2. Signals for protons of the Tyr4, His6, and Phe8 side chains as well as the peptide N-H signals appear in this region. Positive intermolecular organic solvent-peptide NOEs are observed for all protons of the peptide, including all side chains and the R-proton of each residue. The reduced cross-relaxation parameters (σR) characterizing the NOEs are given in Figure 3 (red symbols). A reduced cross-relaxation parameter σR for each proton of [val5]angiotensin was calculated for 10 conformations consistent with observed intramolecular NOEs in this solvent. These 1714

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Figure 3. Comparison of observed (red) and calculated (blue) initial slopes of NOE vs mixing time curves for protons of [val5]angiotensin II in 35% acetonitrile/water v/v at 0 °C when the acetonitrile (CH3) magnetization is inverted. Experimental results are represented by red squares while the red bars indicate (25% experimental uncertainty. The blue squares represent the average of initial slopes calculated for 10 structures that satisfy the observed intramolecular 1H-1H distance constraints while the blue bars show the range of the calculated initial slopes found for the structures considered. Simulations showed that, under the conditions of the NOE experiment, the initial slopes of NOE vs mixing time curves were good approximations to the corresponding intermolecular cross relaxation parameter σNOE solvent. Multiplets for the following pairs of protons overlap in the proton spectrum of the peptide: His6 H and Phe8 H, Val3 H and Tyr4 H, Arg2 HA and Pro7 HA, and Tyr4 HA and Phe8 HA. The NOE observed for coincident signals is plotted at both positions in each case. An experimental result for His6 HA is not shown since this multiplet is close enough to the position of the water resonance that suppression of the water signal distorts its intensity.

calculations utilized the experimental translational diffusion coefficients (Table 1) and the rotational correlations times τR = 0.6 ns and τM = 0.1 ns. The range of σR values calculated for these structures is shown in Figure 3 (blue symbols). These hard sphere calculations for acetonitrile-peptide interactions predict a positive σR for all protons of the peptide and show that σR is sensitive to peptide conformation. 33% Acetone at 0 °C. An intermolecular NOE spectrum of [val5]angiotensin II dissolved in 33% acetone/water (v/v) is shown in Figure 4. Figure 5 compares experimental σR data to reduced initial slopes calculated for the 10 structures consistent with observed intramolecular 1H-1H NOEs (given in the Supporting Information). Comparing calculated and observed peptide intramolecular spin-lattice relaxation rates gave values of τR and τM of 0.8 and 0.08 ns, respectively, for this system. A striking aspect of the intermolecular NOE data in this solvent mixture is the small cross-relaxation terms observed for the peptide N-H protons. NOEs for the peptide backbone CRH protons were positive and somewhat larger. All observable sidechain protons appearing in the upfield portion of the [val5]angiotensin spectrum had positive NOEs. The chemical shift of the Pro7 Hγ2 proton is very close to the acetone resonance. Consequently, it was not possible to invert the acetone magnetization without perturbing to some extent this peptide proton. Simulations of the NOE experiments indicated that the initial rate behaviors of the Pro7 Hβ and Hγ protons in intermolecular NOE experiments did not primarily reflect solvent interactions and predicted that intramolecular relaxation due to Pro7 Hγ2 could have some influence on the initial slope for the Phe8 N-H proton. 36% Dimethyl Sulfoxide-Water at 0 °C. The low-field portion of an NOE spectrum of [val5]angiotensin II in 36% dimethyl sulfoxide/water (v/v) is shown in Figure 6. Except for the aromatic ring protons of the Phe8 residue, negative intermolecular organic solvent-peptide NOEs are observed for all protons of the peptide, including the R-proton of each residue. The reduced cross-relaxation parameters (σR) characterizing NOEs are shown in Figure 7 (red symbols). Values of σR for each proton of [val5]angiotensin were calculated for 10 conformations consistent with observed

Figure 4. Intermolecular NOEs on low-field signals of [val5]angiotensin resulting from inversion of the solvent methyl resonance in a mixture of 33% acetone-water (v/v) at 0 °C (top). A control spectrum is shown at the bottom. The mixing time for the NOE experiment was 400 ms. NOEs on the upfield signals of the peptide were positive.

intramolecular NOEs. The calculations used the rotational correlations times τR = 0.6 ns and τM = 0.1 ns. All σR, in all conformations, were predicted to be negative. Water-Organic Solvent Cross Relaxation. All of the intermolecular NOEs described here were produced by inversion of magnetization associated with spins of the organic cosolvent. There is a large concentration of water protons in contact with the organic cosolvent in all samples. [Val5]angiotensin is surely solvated by water to some extent in the mixed solvents studied and it could be argued that an Overhauser effect produced by inversion of the organic solvent resonance is an indirect effect. That is, inversion of the cosolvent magnetization results in a change in the polarization of the water magnetization, which then perturbs the magnetizations of the peptide spins. This possibility was explored by determining water proton-organic cosolvent proton NOEs. As an example, experimental values for σNOE solvent in 1715

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Figure 5. Comparison of observed (red) and calculated (blue) initial slopes of NOE vs mixing time curves for the indicated protons of [val5]angiotensin II in 33% acetone/water (v/v) at 0 °C when the acetone (CH3) magnetization is inverted. Experimental results are represented by red squares, with the red bars indicating (25% experimental uncertainty. The blue squares represent the average of calculated initial slopes for 10 structures that satisfied the observed intramolecular 1H-1H distance constraints. The blue bars show the range of the calculated initial slopes for the 10 structures considered. The experimental NOEs for the peptide N-H protons were very small and the error bars for these were set at (50%. The multiplets for Tyr4 HA and Phe8 HA overlap in the proton spectrum of the peptide. The observed NOE for the coincident signals is plotted at both positions in the figure. Data for Pro7 HA and Phe8 H are not plotted. Due to near-identity of their chemical shifts, inversion of the magnetization of the solvent methyl protons has an influence on the HB2 proton of Pro7. Pro7 HA and Phe8 H are physically proximate to Pro HB2 in all conformations; calculations indicate that changes in their multiplets during the intermolecular NOE experiment cannot accurately reflect the solvent-based NOE for either Pro7 HA or Phe8 H.

magnetization of the more abundant water spins to produce the intermolecular NOEs observed in this work.

Figure 6. Intermolecular NOEs on low-field signals of [val5]angiotensin resulting from inversion of the solvent methyl resonance in a mixture of 36% dimethyl sulfoxide-water (v/v) at 0 °C (top). A control spectrum is shown at the bottom. The mixing time for the NOE experiment was 300 ms. NOEs on all upfield signals of the peptide were negative, while the NOE on the TSP reference signal was positive.

35% acetonitrile-water were 0.012 and 0.034 s-1 when inverting the acetonitrile methyl or water resonance, respectively. Such data shows that, were an indirect process operating, the water magnetization would be changed by only a few percent through cross relaxation with the organic solvent component over the course of the mixing times used in the intermolecular NOE experiments. Calculations of σR showed that the influence of the indirect mechanism in producing an acetonitrile-peptide proton NOE would be undetectable. Even with solvent-solvent cross-relaxation terms 10 times more efficient than the experimental values, the effects predicted were less than 0.1% of the observed σR. Thus, there is no support for the notion that perturbing the magnetization of the organic cosolvent alters the

’ DISCUSSION The three-dimensional structure of angiotensin, especially as it interacts with its receptors, has been of continuing interest in the context of drug development.43-45 When bound to one monoclonal antibody, the peptide backbone takes up a C shape which brings the N- and C-terminals to within ∼7 Å of each other,46 although crystallographic study of another antibodyangiotensin complex showed the peptide in a nearly fully extended conformation, with peptide termini separated by about 18 Å (Pan et al., in RCSB PDB Protein Databank structure 3CK0). There is evidence that angiotensin II takes up a single, dominant conformation in nonpolar media;47 NMR studies of angiotensin II in water at 5 °C suggested a conformation for the hormone that is similar to the conformation of the peptide when bound to a highaffinity antibody.48 We were able to obtain relatively few NOE and ROE distance constraints for [val5]angiotensin and could only conclude that this analogue in the organic solvent-water mixtures used for the present work is likely present as an equilibrating collection of conformations,49 some of which are largely extended and others somewhat folded. Indicators of the extended conformations include the JNHCRH coupling constants which approach the values expected for the β-sheet conformation while detection of some medium range NOEs are suggestive of more folded conformations. Under these conditions, all NMR observations, including intra- and intermolecular NOEs, are characterized by parameters that are weighted averages of parameters for each conformation present. The observed hydrodynamic radius for the peptide under our experimental conditions (∼12 Å) is larger than the radii expected for the conformations we found to be consistent with the observed 1H-1H NOEs (∼7.5 Å) and is also larger that the experimental value for the angiotensin in 4 M urea, presumably present as a random coil (9.1 Å).50 The 2-4 Å increase in radius observed in the mixed organic-water solvents at 0 °C is suggestive of solvent interactions with [val5]angiotensin that 1716

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Figure 7. Comparison of observed (red) and calculated (blue) initial slopes of NOE vs mixing time curves for the indicated protons of [val5]angiotensin II in 36% dimethyl sulfoxide/water (v/v) at 0 °C when the dimethyl sulfoxide methyl magnetization is inverted. Experimental results are represented by red squares, with the red bars indicating (25% experimental uncertainty. The blue squares represent the average of calculated initial slopes for 10 structures that satisfy the observed intramolecular 1H-1H distance constraints, with the blue bars showing the range of the calculated initial slopes found for the structures considered. Simulations showed that, under the conditions of the NOE experiment, the initial slopes of NOE vs mixing time curves were good approximations to the corresponding intermolecular cross-relaxation parameter σNOE solvent. Multiplets for the following pairs of peptide spins are overlapped in the proton spectrum: His6 H and Phe8 H, Tyr4 HA and Phe8 HA, and Asp2 HA and Pro7 HA. The observed NOE for coincident signals is plotted at both positions. An experimental result for His6 HA is not shown since this multiplet is close enough to the position of the water resonance that suppression of the water signal distorts its intensity.

are sufficiently enduring that the hydrodynamic behavior of the peptide is affected. Peptide N-H chemical shifts provide indications that the organic component of these mixed solvents may interact directly with the peptide. The corresponding N-H shifts for any residue in [val5]angiotensin are similar when cosolvents capable of participating in a hydrogen bond (acetone, DMSO, methanol,21 ethanol,34 ethylene glycol21) are used. Residue N-H shifts are on average 0.29 ppm to higher field when acetonitrile is the cosolvent. Participation in hydrogen bond formation leads to deshielding and the observed N-H shifts in the collection of five solvents mentioned are consistent with peptide-organic solvent interactions in these systems though hydrogen bonding. Strong hydrogen bonding with acetonitrile is not anticipated, leading to the upfield N-H shifts observed when acetonitrile is the cosolvent. Interestingly, only in 35% acetonitrile-water are the observed solvent-peptide cross-relaxation terms uniformly in agreement with those predicted by the hard spheres model (Figure 3), supporting the conclusion that special peptideacetonitrile interactions are absent in this system. The blue bars in Figures 3, 5, and 7 indicate the sensitivity of organic cosolvent-peptide cross relaxation to peptide conformation. We cannot guarantee that the conformational space of [val5]angiotensin has been sufficiently sampled in our attempts to define this sensitivity. However, calculations of σNOE solvent done with the peptide in a fully R-helical conformation, in a completely extended β structure or in the conformations observed for angiotensin bound to antibodies generally gave predicted cross-relaxation terms for each peptide proton within the ranges indicated in these figures. A rotational correlation time (τR) of 2-3 ns is expected for a rigid sphere of radius ∼12 Å at 0 °C in the solvent mixtures studied. The observed or effective τR (0.6-0.8 ns) derived from consideration of proton spin-lattice relaxation rates for the peptide is appreciably smaller than this, presumably as a result of rapid internal motions of the peptide.51 Calculations showed that 1H-1H spin diffusion within the network of protons defined by the structure of [val5]angiotensin would be rapid if τR ∼ 2 ns is obtained and, contrary to observations, would lead to cross-relaxation

rates that were identical for all protons of the peptide. Calculations indicated that, with the τR ∼ 0.7 ns, spin diffusion effects would be negligible and that initial rates of change of a NOE with mixing time would give a reliable indication of the corresponding solvent-solute cross-relaxation parameter. The validity of conclusions drawn from the cross relaxation data reported here critically depend on the reliability of predictions for the cross-relaxation parameters. The model used for these calculations makes a number of assumptions, including (1) solvent-solute interactions involve hard spheres, (2) the composition of the solvent mixture is homogeneous throughout the sample, (3) the mutual diffusion of solvent and solute is adequately represented by experimental (bulk) translational diffusion coefficients, and (4) solvent molecules rotate rapidly under all conditions. Despite the crudeness of this model, crossrelaxation terms calculated for the TSP reference compound agreed reasonably well with experimental results in all solvent systems (Figures 3, 5, and 7). Observed and calculated σR for acetonitrile-peptide interactions agree when the conformational sensitivity of this parameter and experimental errors are considered (Figure 3). In 35% acetone-water and 36% DMSO-water, many experimental cross-relaxation terms are similar to those predicted using this model. Thus, several known aspects of mixed organic-water solvents appear to have little influence on cross-relaxation terms, including the microscopic heterogeneity of acetonitrile-water,52 acetone-water,53 and dimethyl sulfoxide-water53,54 mixtures, and the ability of one component of a mixed solvent to accumulate preferentially near parts of a solute structure.11,55 However, some large disagreements between experimental and predicted σR cross relaxation terms are apparent in the acetone-water and dimethyl sulfoxide-water systems. These likely signal peculiarities of local peptide-organic solvent interactions. In dimethyl sulfoxide-water, σR is predicted to be relatively small and negative for the aromatic ring protons of Phe8, in contrast to the observed positive σR (Figure 6). Within the context of the hard spheres model, the sign of σR depends only on the value of τ, with a small value of τ associated with a positive σR. If DMSO molecules near the peptide move in such a 1717

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The Journal of Physical Chemistry B way that the hydrogens of the solvent preferentially orient on average ∼0.5 Å closer to the Phe8 aromatic ring protons than the center of the sphere representing the solvent molecule, σR would be positive and of the magnitude observed. Such a preferential orientation of the solvent molecules could be consistent with hydrophobic contact of the DMSO methyl groups and the aromatic ring. A similar interaction would also bring the observed and calculated cross-relaxation parameters for the hydrophobic methyl groups of the Val3 and Val5 residues into better agreement. Alternatively, a local increase in the mutual diffusion coefficient (D) could lead to a reduction of τ and could account for the larger than expected σR values for the Phe8 aromatic protons and the valine methyls. Mass spectrometric studies have provided evidence that phenol is selectively solvated by DMSO molecules in 36% DMSO-water.54 Experimental cross-relaxation terms for DMSO interacting with the side-chain protons of the Tyr4 residue of [val5]angiotensin appear to be reliably predicted by the hard spheres model used (Figure 7) and selective accumulation of DMSO near this residue need not be invoked to explain the observed cross-relaxation parameters for the Tyr4 side chain. There is a significant disagreement between observed and calculated σR for the Val3 N-H proton of [val5]angiotensin in DMSO-water (Figure 7). Although the hard spheres model predicts the sign of σR to be negative, in agreement with observation, the magnitude of the experimental value is over 3 times larger than the predicted value. This difference is likely not the result of selective accumulation of DMSO molecules near the Val3 residue: a change from 36% DMSO to 100% DMSO could at most produce a change in the cross-relaxation parameter of a factor of 2.8. We could find no adjustment of r or D in the hard spheres model used to predict σR that would produce a change of the observed magnitude. This part of the peptide may be structured so that associations of DMSO molecules persist long enough that the dipolar interactions of solvent and solute protons become more like intramolecular interactions. Such interactions have been postulated to be consistent with observed solvent-[val5]angiotensin cross-relaxation terms in ethanolwater34 and trifluoroethanol-water33 solvent mixtures at 0 °C. Although the data are more scattered, observed and calculated cross-relaxation terms for the side chains of the peptide in 35% acetone-water are in reasonably good agreement. Cross-relaxation terms near zero are observed for the backbone N-H protons while negative σR are predicted. Reminiscent of the DMSO-water results, if acetone molecules near the surface of the peptide on average tend to be preferentially oriented so that the acetone methyl protons are ∼1.3 Å closer to an N-H proton than is the center of the sphere representing the acetone, better agreement between observed and calculated σR is obtained. But, again, the same result can be produced by adjusting the local value of D.

’ SUMMARY Intermolecular NOEs have been used to examine the interactions between three dipolar aprotic cosolvents in water with the peptide hormone [val5]angiotensin. The solvent mixtures used were 35% acetonitrile-water (v/v), 33% acetone-water (v/v), and 36% dimethyl sulfoxide-water (v/v). Observed cross-relaxation terms were considered in light of predictions of a model for spin dipolar interactions in which solvent and solute interact as hard spheres encountering each other by mutual diffusion.

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The predictions used experimental translational diffusion coefficients and take into account the conformation of the peptide, but ignore other aspects of organic solvent-water mixtures, particularly microheterogeneity. Intramolecular 1H-1H NOE data showed that the hormone does not have a single dominant conformation in these solvents but appears to be a mixture of conformations, some of which may be somewhat folded. Predictions of solvent-peptide cross relaxation terms took into account the range of conformations indicated by the intramolecular NOE data. In many cases, the observed organic solvent-peptide dipolar cross relaxation terms agree with those predicted by the hard spheres model. However, the experiments signaled unusual solvent interactions at the Val3 and Phe8 residues in dimethyl sulfoxide-water and at all peptide N-H protons and the Tyr4 side chain in acetone-water. Plausible mechanisms for producing these effects are suggested but the current experiments cannot definitively establish any of these.

’ ASSOCIATED CONTENT

bS

Supporting Information. Chemical shift assignments for all solvent systems, vicinal coupling constants, indications of the number and distribution of observed 1H-1H intramolecular NOEs of the peptide in each solvent system, and further discussion of the methods used to obtain the solvent-solute cross-relaxation terms responsible for the observed intermolecular NOEs. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected]. Tel.: 805-893-2113. Fax: 805-8934120.

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